Aspergillus niger absorbs copper and zinc from swine ...

Bioresource Technology 77 (2001) 41±49

Aspergillus niger absorbs copper and zinc from swine wastewater Michael S. Price a, John J. Classen b, Gary A. Payne a,* b

a Department of Plant Pathology, Box 7616, North Carolina State University, Raleigh, NC 27695-7616, USA Department of Biological and Agricultural Engineering, Box 7625, North Carolina State University, Raleigh, NC 27695-7625, USA

Received 18 January 2000; received in revised form 8 August 2000; accepted 1 September 2000

Abstract Wastewater from swine con®ned-housing operations contains elevated levels of copper and zinc due to their abundance in feed. These metals may accumulate to phytotoxic levels in some agricultural soils of North Carolina due to land application of treated swine euent. We evaluated fungi for their ability to remove these metals from wastewater and found Aspergillus niger best suited for this purpose. A. niger was able to grow on plates amended with copper at a level ®ve times that inhibitory to the growth of Saccharomyces cerevisiae. We also found evidence for internal absorption as the mechanism used by A. niger to detoxify its environment of copper, a property of the fungus that has not been previously exploited for metal bioremediation. In this report, we show that A. niger is capable of removing 91% of the copper and 70% of the zinc from treated swine euent. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Copper; Zinc; Bioremediation; Wastewater; Absorption; Fungi; Aspergillus niger

1. Introduction Fungi are used widely in biotechnology for many processes, including the production of antibiotics, enzymes, food products, industrial acids, and alcohol. Fungi also have been utilized for removal of eutrophication agents and bioremediation of metal contaminated waste streams (Thanh and Simard, 1973; Akthar and Ghaar, 1986; Akthar and Mohan, 1995; Bosshard et al., 1996). There is current interest in the use of microorganisms for the removal of nitrogen, phosphorus, and metals from commercial and municipal waste (Cassidy et al., 1996). North Carolina is currently the second largest pork producing state in the US, with an on-farm inventory of 9.5 million swine (USDA, 1999). Nationwide, increased pork production has been accompanied by changes to modern farming methods, including con®nement housing and higher population densities. These changes have led to larger volumes of waste being generated on smaller areas of land. While other research is targeting carbon, nitrogen, and phosphorus removal from

*

Corresponding author. Tel.: +919-515-6994; fax: +919-515-7716. E-mail address: [email protected] (G.A. Payne).

wastewater, copper and zinc removal has been neglected. Copper and zinc salts are liberally included in swine feed as additives. These metals promote immune system function and growth in swine (Omole, 1980; O'Dell, 1998), are inexpensive, and are relatively nontoxic to hogs, at doses greater than that required for maintenance, due to their lower bioavailability (O'Dell, 1998). The unabsorbed copper and zinc in swine feed are excreted. Over time, these metals could accumulate to phytotoxic levels in soil receiving swine manure or wastewater. Primary swine waste treatment systems target carbon stabilization. Current land application disposal systems are based on a balance of nitrogen application and uptake by plants. New systems under investigation attempt to enhance nitrogen and phosphorous removal from liquid euent in order to reduce the amount of land required for ®nal disposal. These enhanced nutrient removal systems would, therefore, raise the amount of regulated metals applied to a given area of land by allowing higher concentrations of waste to be applied. Industrial methods to remove these metals, such as ion exchange resins, are inappropriate for use in animal agriculture due to the high cost of implementing these systems and their non-speci®c metal binding properties. An ideal system for the removal of regulated metals, such as copper and zinc, would be one

0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 3 5 - 8

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M.S. Price et al. / Bioresource Technology 77 (2001) 41±49

that is economical and safe for the environment, as well as ecient in removing target metals. One fungus shown capable of removing metals from a number of substrates is Aspergillus niger. This ®lamentous fungus is widely used in the fermentation industry because of its ease of culturing and lack of pathogenicity to humans and animals (Berka et al., 1992). A. niger has been used to remove metals from the environment by either adsorption of the metals to fungal cell wall components, or complexation of the metals with organic acids produced by the fungus (Akthar and Mohan, 1995; Bosshard et al., 1996). Other fungi also have been used for metal removal with varying degrees of success (de Rome and Gadd, 1987; Zhou and Ki, 1991; Mullen et al., 1992; Brady and Duncan, 1994; Al-Asheh and Duvnjak, 1995; Zhang et al., 1998). Speci®cally, Fusarium species have been used as biosorbents, and Penicillium simplicissimum has been used to precipitate metals from solution (Burgstaller and Schinner, 1993; Gadd, 1993; White et al., 1997). However, A. niger has been consistently listed among the top metal biosorbents (Luef et al., 1991; Mullen et al., 1992; Gadd, 1993; Akthar and Mohan, 1995; Bosshard et al., 1996). In this study, we evaluated six fungi for their ability to grow on and remove copper and zinc from culture media and swine wastewater. The objective of this study was to evaluate potential fungi for use in bioremediation of copper and zinc from swine wastewater. Further, we wanted to determine if current bioremediation protocols for wastewater metal removal utilize fungi in the most ecient manner. Of the fungi tested in an initial screen, A. niger was the most eective at removing copper from liquid media. Its ability to remove copper and zinc from treated swine wastewater was further studied. 2. Methods 2.1. Microorganisms used Aspergillus niger (NRRL 326) and Penicillium simplicissimum (NRRL 1075) were obtained from the USDA/ARS National Center for Agricultural Utilization Research, Peoria, IL. F. verticillioides (Gibberella fujikuroi strain A00149) was obtained from the Fungal Genetics Stock Center, Kansas State University. Rhizoctonia solani and Aquathanatephorus pendulus isolates were obtained from Dr. Marc Cubeta, North Carolina State University (NCSU). The Geotrichum isolate was collected from soil adjacent to the Lake Wheeler Road Field Laboratory Swine Educational Unit lagoon, Wake County, NC. All fungi were cultured on potato dextrose agar for production of conidia, or in case of R. solani and A. pendulus, for mycelium. All liquid cultures in the metal uptake and detoxication mechanism studies were carried out in duplicate.

2.2. Fungal growth screen The fungi were grown for 7 days at 28°C on either yeast extract/sucrose (YES) agar (yeast extract, 2%; sucrose, 5±10%; Bactoâ agar, 15 g/l), YES 5 mM CuSO4 5H2 O, or a minimal medium of the following composition (g/l): L -asparagine, 0.5; K2 HPO4 ; 0:5; MgSO4 7H2 O; 0:2; FeSO4 7H2 O; 0:01; D -glucose, 2.0; Bactoâ agar, 15. The center of plates containing the respective media was inoculated with 5 ll of 0.05% Triton X-100 containing 5000 spores of either A. niger, F. verticillioides, P. simplicissimum, or the Geotrichum sp. isolate. A 3.5 mm plug from the margin of growing colonies was used to inoculate plates with R. solani and A. pendulus. The diameter of the mycelial plug approximated the diameter of the spore suspension droplet applied to the media. Three plates of each fungus were used for each treatment. The mean of perpendicular diameter measurements was recorded for each plate on day 7. 2.3. Copper uptake screen To determine the relative ability of the fungi tested to remove copper from liquid media, the fungi were cultured in 100 ml YES broth amended with 0.1 mM CuSO4 . The medium was inoculated with 106 conidia/ml for all fungi except R. solani and A. pendulus. For these fungi, ten 3.5 mm plugs were added to each ¯ask. All cultures were incubated at 28°C for 4 days. The fungal biomass were then washed for 30 min in 0.1 N HCl. Because F. verticillioides and Geotrichum sp. grew as conidia or short mycelial strands, the washes from these fungi were separated by centrifugation at 10; 000 g for 30 min. The biomass of all other cultures was separated by ®ltration through P5 Whatman paper. All fungal biomass samples were lyophilized. The spent broths, wash ®ltrates, and lyophilized biomasses were analyzed for copper content. 2.4. Determination of metal removal mechanism(s) in A. niger A. niger was further assayed for its ability to remove metal from YES broth amended with 0.0, 0.01, 0.1 or 1.0 mM CuSO4 5H2 O or ZnSO4 7H2 O. The +2 valence form of both metals has been used in other studies (Junghans and Straube, 1991; Luef et al., 1991; Kermasha et al., 1993; Al-Asheh and Duvnjak, 1995; Morley and Gadd, 1995; Sanchez et al., 1999). In further studies to assess possible interaction between copper and zinc uptake, both metals were present at equal concentrations at the levels listed above. Also, titration experiments were designed with each metal titrated against the other. For example, copper was ®xed at 30 lM (the concentration of copper in fresh waste), while zinc was


varied over a range from 50 to 500 lM (a range including the concentration of zinc in fresh waste). The fungus was grown at 28°C, harvested, and the biomass and ®ltrates prepared for analysis as described above. To determine if uptake was due to an active or passive process, the removal of copper from a 0.1 mM solution of CuSO4 by live and killed cultures of A. niger was compared. A. niger spores (105 spores/ml) were inoculated into two ¯asks containing 1 l each of a modi®ed Watson and Smith medium (Kermasha et al., 1993) and incubated as shake cultures (125 rpm) for 4 days at 28°C. The two cultures were then harvested by ®ltration through P5 Whatman paper and the mycelial biomass of each ¯ask was divided into halves. Each half was further cut into squares approximately 1 cm2 . Squares from half of each culture were then killed by exposure to chloroform vapor for 20 min. All of the diced halves were then weighed and inoculated into ¯asks (three replicates per treatment) containing 100 ml each of the Watson and Smith broth with 0.1 mM CuSO4 and incubated at 28°C for 24 h as shake cultures (125 rpm). The cultures were then harvested, and the biomass and ®ltrates were prepared for analysis as described above. 2.5. Metal removal from swine wastewater For the evaluation of metal removal from treated swine wastewater, A. niger was grown in waste liquid obtained from the Center for Environmental Farming Systems (CEFS), Goldsboro, NC, operated by the North Carolina Department of Agriculture. Waste samples were obtained from the house ¯ush tank containing euent from this alternative treatment system. The CEFS treatment system uses aerobic and anoxic tanks in alternating sequence to remove nitrogen by nitri®cation and denitri®cation. Seed cultures were grown to approximately 1.0 g dry weight in 100 ml YES broth for 48 h at 28°C, and resuspended in 100 ml of treated euent. One seed culture was used for each ef¯uent culture. All euent cultures were incubated at 28°C in a rotary shaker incubator (150 rpm) for 24 h. At the end of the incubation, the biomasses were removed by ®ltration as described above and the liquids were centrifuged at 10; 000 g for 30 min to remove any organic debris. The mycelium retained on the ®lters was washed with sterile 0.05% Tween-20 to remove any microbes associated with the fungal cell walls. The wash ®ltrates were collected by ®ltration through P5 Whatman paper and the fungal biomasses were lyophilized. The supernatants, pellets, wash ®ltrates, and fungal biomasses were all analyzed for copper and zinc content. 2.6. Sample analysis All samples were analyzed by atomic absorption spectroscopy (Clesceri et al., 1990) at the Biological and

43

Agricultural Engineering Department's Environmental Analysis Laboratory, NCSU. Filtered fungal biomass and centrifuged pellets were lyophilized and weighed prior to analysis. All glassware used in these experiments was rinsed with a solution of 25% ACS grade nitric acid to remove any metal contaminants. Statistical analysis was performed on data using the Microsoft Excel 97/98 software package (Microsoft, Redmond, WA). 3. Results 3.1. Fungal growth screen All of the fungi tested except R. solani were able to grow in the presence of 5.0 mM CuSO4 (Fig. 1). A. niger, however, grew considerably better on copper-amended medium than any other fungus and obtained a colony diameter of 84.5 mm in 7 days (Fig. 1). Further, the morphology of A. niger grown on copper-amended medium did not appear dierent from that observed on control YES medium. The fungus grew luxuriantly on the copper-containing medium and produced abundant conidia. In contrast, the presence of 5.0 mM copper in the medium caused observable changes in the morphology of the other fungal species tested. For example, A. pendulus, which obtained a colony diameter nearly as large as A. niger on copper-containing media, produced only sparse mycelial growth. 3.2. Copper uptake screen Five of the six fungal species were further tested for their ability to remove copper from broth solution amended with 0.1 mM CuSO4 . The amount removed varied from 0.09 (A. pendulus) to 0.52 (A. niger) mg copper/g fungal dry weight. A. niger not only grew better than the other fungi (3.1 g dry weight), but it also removed more copper per gram fungal dry weight than did the other fungi (Fig. 2). Geotrichum and F. verticillioides removed 0.41 and 0.29 mg copper/g dry weight, respectively, and grew to 1.1 and 1.0 g dry weight, respectively. In the solution, both Geotrichum and F. verticillioides grew as conidia or short mycelial strands, whereas the other fungi grew as mycelium. 3.3. Mechanism of copper and zinc removal by A.niger We chose A. niger for further study because of its ability to eciently remove copper from solution. As our initial interest was to use a fungus to remediate swine lagoon wastewater, we evaluated A. niger for its ability to grow in and to remove copper and zinc from swine lagoon wastewater. Swine waste samples from a representative lagoon contained 0.11 mM copper as well as 0.037 mM zinc. To begin our comparative studies of

44


Fig. 1. Growth of six fungi on solid media with or without 5.0 mM CuSO4 . Fungi were grown at 28°C for seven days on YES , minimal medium (MM) , or YES 5:0 mM CuSO4 j. Bars represent standard error among three replicates. An Aspergillus niger, Aq Aquathanatephorus pendulus, Ge Geotrichum sp., Fv Fusarium verticillioides, Ps Penicillium simplicissimum, Rs Rhizoctonia solani.

Fig. 2. Amount of copper removed from a 0.1 mM solution of CuSO4 by various fungal species. Fungi were grown at 28°C for 4 days in YES broth supplemented with 0.1 mM CuSO4 . Abbreviations same as used in Fig. 1. Values represent the means from two replicated experiments.

copper and zinc uptake by A. niger, we examined the pro®le of copper and zinc accumulation by the fungus. A. niger was grown in shake culture (125 rpm) at 28°C for one week and the biomasses and ®ltrates were assayed daily for metal content. When A. niger was grown in the presence of 0.1 mM copper, approximately the copper concentration found in swine lagoons, removal of copper increased from 58% after 1 day to 99% removal after 7 days (Table 1). Copper removal by the fungus was near maximal by day four (Table 1). Interestingly, the amount of copper removed per gram fungal dry weight decreased over time (Table 1). Thus, the increase in copper removal from solution over time was due to increased mycelial growth, not more ecient copper absorption. The removal of zinc over time showed a similar pro®le to that for copper, except that the absolute amount removed was an order of magnitude less than for copper. Only 10% of the zinc from a 0.05 mM solution (a concentration comparable to that found in

Table 1 Removal of copper and zinc from culture medium by Aspergillus niger Day

1 2 3 4 5 6 7

Culture metal content 0.1 mM copper

0.05 mM zinc

Removed (%)

Removed (mg/g dry wt.)

Removed (%)

Removed (mg/g dry wt.)

0:1mM copper 0:05mM zinc Cu removed (%)

Cu removed (mg/g dry wt.)

Zn removed (%)

Zn removed (mg/g dry wt.)

58.3 68.9 91.4 94.2 98.3 99.1 99.2

2.58 1.08 0.89 0.83 0.72 0.61 0.57

9.8 16.2 13.2 20.9 23.2 30.6 36.9

0.20 0.18 0.13 0.13 0.13 0.15 0.15

60.6 71.8 93.4 96.0 97.2 96.4 98.5

1.63 0.97 1.00 0.85 0.65 0.32 0.53

15.6 22.1 19.3 24.9 33.8 40.1 46.5

0.24 0.21 0.15 0.15 0.19 0.20 0.18


45

swine lagoon wastewater) was removed in the ®rst day of culture. The maximum amount of zinc removed, 37%, occurred after 7 days (Table 1). The amount of zinc removed per gram fungal dry weight was also less than that for copper and ranged from 0.1 to 0.2 mg Zn/g fungal dry weight (Table 1). To determine if the presence of copper and zinc together in solution altered the uptake of either metal, A. niger was cultured in a solution containing 0.1 mM copper and 0.05 mM zinc. The pro®le of copper accumulation was nearly identical to that found when copper was present alone (Table 1). Similarly, the pro®le of zinc removal in the copper/zinc solution mimicked that found when zinc was present alone (Table 1). In order to determine if the uptake of copper and zinc was concentration dependent, A. niger was further tested for its ability to remove these metals from medium containing 0.01, 0.1 or 1.0 mM CuSO4 or ZnSO4 . In these studies, the mycelial biomasses were treated with 0.1 N HCl after harvest and the concentration of copper or zinc determined in the medium, in the HCl wash and in the mycelial biomass. Treatment of the fungal mycelium with 0.1 N HCl has been reported to remove any copper adsorbed to the mycelium (Gadd, 1993; Akthar and Mohan, 1995). In our studies, all copper and zinc remaining with the biomass after this treatment was assumed to be absorbed internally. As shown in Fig. 3, the amount of copper removed from solution (absorbed and adsorbed) increased linearly (on a log±log plot) in response to increased copper concentrations. A. niger was capable of removing up to 3.65 mg copper/g fungal dry weight from a 1.0 mM solution of CuSO4 . In 0.01 mM copper, the fungus removed approximately 79% of the copper present (0.044 mg copper/g fungal dry weight), with 73% by absorption and 6% by adsorption. The fungus absorbed

88.5% (1.43 mg) and adsorbed 9% (0.15 mg) of the copper from a 0.1 mM CuSO4 solution, which corresponded to 0.518 mg copper/g fungal dry weight. Thus A. niger was able to remove 97% of the copper from a solution containing approximately the same concentration of copper found in swine lagoon wastewater (0.11 mM). To determine if copper removal was accomplished by an active or passive process (i.e., binding fungal cell walls), copper removal by living and chloroform-killed biomass was compared. As shown in Fig. 4, the living fungal tissue absorbed ®ve times more copper (on a mass basis) than a metabolically inactive biomass. The living fungal biomass removed 82% of the total copper present in the medium while the killed material removed only 7%. A. niger was grown in solutions containing ZnSO4 at the same concentrations used for the copper study to determine if it could also remove zinc from solution. As shown in Fig. 5, A. niger removed 0.533 mg zinc/g fungal dry weight from a 1.0 mM solution of ZnSO4 . Zinc, like copper, was both absorbed and adsorbed by the fungus. In a 0.01 mM solution of ZnSO4 , A. niger removed 0.164 mg zinc/g fungal dry weight, which corresponded to 66% absorption of the total zinc present. At 0.1 mM zinc, the concentration nearest that found in swine wastewater (0.194 mM in treated swine euent), A. niger was able to remove 0.37 mg zinc/g fungal dry weight (Fig. 5). The 40.1% of zinc removed was almost evenly divided between absorption (20.4%) and adsorption (19.7%). Copper and zinc did not aect the removal of oneanother from a solution containing 0.1 mM copper and 0.05 mM zinc (Table 1). A possible interaction between the two metals was further examined by measuring the uptake of each metal from a solution containing equal molar concentrations of copper and zinc in a range from 0.01 to 1.0 mM. The weight of each metal removed

Fig. 3. Copper accumulation by Aspergillus niger grown in dierent concentrations of CuSO4 . A. niger was grown in YES broth amended with CuSO4 at the concentrations shown, and was grown in shake ¯asks (150 rpm) at 28°C for 7 days. Values represent the means from two replicated experiments.

Fig. 4. Eect of metabolic status on copper removal by Aspergillus niger. A. niger was grown at 28°C for four days in a modi®ed Watson and Smith medium in shake ¯asks at 125 rpm. At this time half the biomass was killed with chloroform vapor, and the live and dead biomasses were challenged separately with 0.1 mM CuSO4 in shake culture for 24 h. Bars represent standard error among three replicates.

46


its ability to remove the two metals from swine euent. We assayed treated swine euent by atomic absorption spectroscopy and found copper present at 0.029 mM and zinc at 0.194 mM. The fungus was able to remove 91% of the copper and 70% of the zinc from treated swine wastewater after 24 h incubation of the fungal biomass (data not shown). These percentages correspond to 0.05 and 0.96 mg metal/g fungal dry weight of copper and zinc, respectively, being removed by the fungus. 3.5. Eect of varying the metal concentration on the uptake of the other metal

The observation that A. niger can remove both copper and zinc from de®ned media encouraged us to study

The amount of zinc removed from swine wastewater was higher than that observed when the fungus was grown in culture media amended with zinc or copper alone (data not shown, Table 1). One possible explanation for this is that, copper facilitates zinc uptake at the zinc concentration present in wastewater. To test this hypothesis, solutions were made in which zinc concentration was held constant at 180 lM and the concentration of copper varied from 1±50 lM. As can be seen in Fig. 7, copper concentration in the range of that found in swine wastewater did not aect zinc removal from solution. Increasing the concentration of copper did result in increased copper uptake as would be expected. The eect of diering zinc concentrations on copper uptake was also examined. A. niger was grown in solutions in which zinc was increased from 10 to 500 lM with a constant copper concentration of 30 lM. Increasing zinc concentration in this range increased zinc removal by the fungus but did not aect copper removal from the media (Fig. 8).

Fig. 6. Accumulation of copper and zinc by Aspergillus niger grown in a medium containing both metals. A. niger was grown at 28°C for seven days in YES broth amended with 0.01, 0.1, or 1.0 mM each of CuSO4 and ZnSO4 . Spent broth, 0.1 N HCl wash ®ltrate, and fungal mat samples were analyzed for metal content by atomic absorption spectroscopy. Removed metal is de®ned as the metal associated with the wash and mat. Values represent the means from two replicated experiments.

Fig. 7. Titration of copper against a constant level of zinc and its eect on metal uptake by Aspergillus niger. A. niger was grown at 28°C for four days in a modi®ed Watson and Smith medium in shake ¯asks at 150 rpm. Zinc concentration held constant at 189 lM as ZnSO4 , and copper concentration varied at either 1, 5, 10 or 50 lM as CuSO4 . Bars represent standard error among three replicates.

Fig. 5. Zinc accumulation by Aspergillus niger grown in dierent concentrations of ZuSO4 . A. niger was grown in YES broth amended with ZnSO4 at the concentrations shown, and was grown in shake ¯asks (150 rpm) at 28°C for 7 days. Values represent the means from two replicated experiments.

(mg/g fungal dry weight) when present together at either 0.01 or 1.0 mM was approximately the same as when each metal was present alone (Figs. 3, 5 and 6). The amount of copper removed at 0.1 mM (when present with zinc) decreased compared to copper alone, from 0.52 to 0.39 mg copper/g fungal dry weight (Figs. 3 and 6). Likewise, zinc removal (when present with copper) decreased compared to zinc alone (0.1 mM), from 0.37 to 0.13 mg zinc/g fungal dry weight (Figs. 5 and 6). 3.4. Metal removal from swine wastewater


Fig. 8. Titration of zinc against a constant level of copper and its eect on metal uptake by Aspergillus niger. A. niger was grown at 28°C for four days in a modi®ed Watson and Smith medium in shake ¯asks at 150 rpm. Copper concentration held constant at 30 lM as CuSO4 , and zinc concentration varied at either 10, 50, 100 or 500 lM as ZnSO4 . Bars represent standard error among three replicates.

4. Discussion Filamentous fungi are used widely in industrial fermentation and bioremediation (Friedrich et al., 1983, 1986; Luef et al., 1991; Berka et al., 1992; Mullen et al., 1992; Burgstaller and Schinner, 1993; Feo®lova et al., 1994; Akthar and Mohan, 1995; Bosshard et al., 1996; Plaza et al., 1996). They are preferred over other organisms for bioremediation because they are easier to remove from liquid substrates. Metal contamination of swine waste is a good example of a system in which bioremediation is ecologically sound. Swine wastewater does not contain high concentrations of metals per se, but the contaminating metals may accumulate to phytotoxic levels in soils receiving repeated applications of swine euent, as is the practice in North Carolina. Our studies indicate that A. niger is capable of thriving on substrates containing elevated levels of copper. It grew better in the presence of copper than all the other fungi tested, to a maximum of 3.1 g dry weight after 4 days in liquid culture containing 0.1 mM copper. Further, this fungus is able to grow normally on plates amended with a level of copper ®ve times greater than that inhibitory to the growth of S. cerevisiae (Fogel and Welch, 1982). Our data suggest a copper concentration greater than 20 mM is required for inhibition of growth (data not shown). We further show that A. niger can accumulate copper and zinc from semi-de®ned media as well as swine wastewater. This fungus is capable of removing up to 0.533 mg Zn and 3.65 mg Cu/g fungal dry weight from semi-de®ned media (Figs. 3 and 5). When assayed in semi-de®ned media with copper and zinc present at the same concentrations found in treated swine euent, A. niger was able to remove only 13% of the zinc and 48% of the copper present (data not

47

shown). In treated swine euent, however, this fungus was able to remove 70% of the zinc and 91% of the copper present (data not shown). There was no clear interaction between copper and zinc that would aect the uptake of these metals from solution by A. niger. When the copper concentration in solution was held constant at 30 lM and zinc concentrations were adjusted from 10 to 500 lM, neither copper nor zinc uptake diered compared to each present in solution alone (Figs. 5 and 7). Similarly, copper uptake was not aected when zinc was held constant at 180 lM and copper concentrations were adjusted from 1 to 50 lM (Figs. 3 and 8). Further, in experiments in which copper and zinc were in solution in equal concentrations of 0.01 and 1.0 mM, no eect was observed (Figs. 3, 5, 6). An exception to this occurred when copper and zinc were present together at 0.1 mM (Fig. 6), in which removal of zinc was less than when zinc was assayed separately (Fig. 5). When present with an equal molar concentration of copper, removal of zinc slipped from 0.367 mg/g fungal dry weight (zinc alone) to 0.126 mg/g fungal dry weight (zinc with copper). One possible explanation for this reduction could be the optimization of the system for copper tracking, as removal of copper as a percentage of total copper present (with zinc) is near the level of removal observed for copper alone. Other researchers have examined A. niger for its ability to remove contaminating metals (Akthar and Mohan, 1995; Bosshard et al., 1996). Akthar and Mohan (1995) used killed mycelium of A. niger to remove copper and zinc from contaminated lake waters. The levels of copper and zinc in these lake waters was comparable to those found in treated swine euent. The killed biosorbent was able to remove 17.02 lg Cu and 912.5 lg Zn/g fungal dry weight from the contaminated lake water. Bosshard et al. (1996) reported the use of A. niger to remove copper, zinc and other metals from incinerated municipal (¯y) ash by organic acid leaching. The fungus is known to produce large quantities of citrate and gluconate, both of which are capable of leaching or precipitating metals out of materials. While this method works well with substrates possessing extremely high metal content (0.11% Cu, 3.1% Zn in ¯y ash), it may not be suitable for use in the swine wastewater system (0.000003% Cu and 0.000018% Zn). Indeed, we found that using similar growth conditions as were used in the ¯y ash study, copper was absorbed by A. niger rather than precipitated. However, this does not discount the potential to utilize organic acid production by this fungus to remediate solid swine waste. Our data support an active process as being responsible, at least in part, for the fungus' ability to remove such large amounts of copper and zinc from solution. A live fungal biomass is able to remove more than twice the amount of copper from solution than a dead

48


biomass (Fig. 4). We found that A. niger removes metals, especially copper, from its environment mainly by absorption. Initial studies in untreated wastewater from the CEFS system showed all of the removed metal associated with the fungus, and no metal associated with the HCl wash. A. niger was able to remove 51.1 lg Cu and 963 lg Zn/g fungal dry weight from treated swine euent (data not shown), which is superior to the killedfungus biosorption method used by Akthar and Mohan (1995). The absorptive properties of A. niger have not been previously reported for use in bioremediation. The actual mechanism(s) which A. niger uses to detoxify metals present in its environment is unknown. Metallothioneins (MTs) are metal binding proteins which have been postulated as responsible for detoxication of a variety of class IIb metals in many dierent species (Lerch, 1980; Jeyaprakash et al., 1991; Mehra and Winge, 1991; Gadd, 1993; Cervantes and GutierrezCorona, 1994; Kosman, 1994; Cizewski-Culotta et al., 1995; Joho et al., 1995; Kelly et al., 1996; RamirezSalgado et al., 1996; Perego and Howell, 1997). A. niger is known to possess a copper metallothionein (CuMT), but neither the protein nor the gene has been sequenced (Kermasha et al., 1993). Other possibilities, such as phytochelatin and superoxide dismutase, cannot be overlooked. In any case, copper resistance in this organism is due to an active process, and not simply due to metal binding passively on the cell wall. The potential for copper removal in swine wastewater was predicted in our semi-de®ned media studies (Table 1). The level of zinc removal, however, was not predicted by any of our other data. Surprisingly, A. niger's ability to absorb zinc was enhanced in the swine wastewater environment. A. niger accumulated 1.68 mg Zn/g fungal dry weight (highest for a given experiment) from the wastewater. The mechanism for increased zinc accumulation is not known. In conclusion, we have shown that the fungus A. niger is capable of removing both copper and zinc from treated swine euent, and that the mechanism of removal appears to be mainly due to an active metabolic process leading to internal absorption of the metal. Bioremediation by internal absorption has not been previously reported in the literature. If the fungus were to be used in a treatment regime, the spent fungus could be replaced with fresh biomass and possibly fed to swine as a dietary supplement for copper and zinc.

Acknowledgements Support for this research was provided by the North Carolina Agricultural Foundation, and the North Carolina Animal & Poultry Waste Management Center project number 1998-0381.

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